The long-term objective of our research is to understand the organization and expression of regulatory and structural gene systems in simple eukaryotic organisms. As a simple unicellular eukaryote, yeast is an excellent model system for studies on gene organization and regulation in higher cells. The methods we have developed over the past 3 years should now enable us to isolate and study any region of the yeast (or other simple eukaryote) genome, including centromeric DNA. Because of the lack of highly repetitive DNA sequences near the centromere of yeast chromosomes, we are able to isolate the centromeric DNA sequences by overlap hybridization techniques. Elucidation of the molecular events occurring during spindle formation and attachment of spindle fibers to chromosomes should now be possible. Our specific goals are: (1) Isolate and characterize all of the DNA sequences between the cdc10 and pgk genetic loci on chromosome III, which must include yeast centromeric DNA. Define the nature of centromeric DNA in terms of nucleotide sequence. (2) Begin studies on the specific association of nuclear proteins with centromeric DNA. (3) Identify yeast replicators or origins of replication on cloned yeast DNA segments, and determine their nucleotide sequences. (4) Define the nature of the high-level yeast promoters for the glycolytic enzyme genes, using the cloned pgk gene as a model system.